Visible Fluorescence Excitation Research Articles

Optical traps induce fluorophore photobleaching by two-photon excitation

Techniques combining optical tweezers with fluorescence microscopy have become increasingly popular. Unfortunately, the high-power, infrared lasers used to create optical traps can have a deleterious effect on dye stability. Previous studies have shown that dye photobleaching is enhanced by absorption of visible fluorescence excitation plus infrared trap photons, a process that can be significantly reduced by minimizing simultaneous exposure to both light sources. Here, we report another photobleaching pathway that results from direct excitation by the trapping laser alone. Our results show that this trap-induced fluorescence loss is a two-photon absorption process, as demonstrated by a quadratic dependence on the intensity of the trapping laser. We further show that, under conditions typical of many trap-based experiments, fluorescence emission of certain fluorophores near the trap focus can drop by 90% within 1 min. We investigate how photostability is affected by the choice of dye molecule, excitation and emission wavelength, and labeled molecule. Finally, we discuss the different photobleaching pathways in combined trap-fluorescence measurements, which guide the selection of optimal dyes and conditions for more robust experimental protocols.

Relaxation dynamics of 3He and 4He clusters and droplets studied using near infrared and visible fluorescence excitation spectroscopy.

The relaxation dynamics of electronically excited 3He and 4He clusters and droplets is investigated using time-correlated near-infrared and visible (NIR/VIS) fluorescence excitation spectroscopy. A rich data set spanning a wide range of cluster and droplet sizes is produced. The spectral features broadly follow the vacuum ultraviolet excitation (VUV) spectra. However, when the NIR/VIS spectra are normalised to the VUV fluorescence, regions with distinctly different cluster size and isotope dependence are identified, enabling deeper insight into the relaxation mechanism. Particle density, location of atomic-like states and their principal quantum number, n, are found to play an important role in the relaxation. For states with n = 3 and higher, only energy within the surface region is transferred to excited atoms which are subsequently ejected from the surface and fluoresce in vacuum. For states with n = 2, energy from the entire region within clusters and droplets is transferred to the surface, leading to the ejection of excited atoms and excimers. Here, the energy is transferred by excitation hopping, which competes with radiative and non-radiative decay, making ejection and NIR/VIS fluorescence inefficient in increasingly larger droplets.

ChemInform Abstract: Intramolecular Excited-State Proton Transfer in Jet-Cooled 2- Substituted 3-Hydroxychromones and Their Water Clusters.

Intramolecular excited‐state proton transfer (ESPT) of 3‐hydroxychromone (3‐HC), 3‐hydroxyflavone (3‐HF) and 2‐(2‐naphthyl)‐3‐hydroxychromone (2‐NHC), and of their water clusters, was investigated in a supersonic expansion. The visible tautomer fluorescence excitation spectra of these compounds exhibit considerably well‐resolved vibrational structures, while no significant uv fluorescence excitation spectrum due to the normal form was observed. The upper limit of the rate constant of the tautomer formation was estimated to be 1.77×101 2 s− 1 for 3‐HC, 6.5×101 1 s− 1 for 3‐HF, and 1.54×101 1 s− 1 for 2‐NHC in supersonic expansion by simulation of the linewidth of the respective origin bands in the visible fluorescence excitation spectra. The order of rate constants of the tautomer formations in the supersonic free jet are consistent with that of the ESPT of these compounds in nonpolar solution. The fluorescence excitation and dispersed fluorescence spectra demonstrate 1:1 and 1:2 water complex formations of 3‐HF and 2‐NHC and no ESPT in these water clusters. However, no evidence of water complex formation was obtained in jet‐cooled 3‐HC.

Analysis and fit of the high-resolution spectrum of the Ã1A u– [formula omitted]1A g LIF spectrum of the two-equivalent-top molecule biacetyl

The jet-cooled laser-induced visible fluorescence excitation spectrum of the à 1 A u (S 1)– X ˜ 1 A g ( S 0) transition in biacetyl (CH 3 C( O) C( O) CH 3) exhibits a long progression in the torsional vibrations of the two equivalent methyl tops in this molecule, whose structure has previously been described and qualitatively understood using local mode ideas applied to the two equivalent methyl rotor torsions together with the G 36 symmetry species A 1, A 2, A 3, A 4, E 1, E 2, E 3, E 4, and G. In the present rotational analysis, we have assigned a G 36 symmetry species, two local-mode torsional quantum numbers, and the usual three asymmetric rotor quantum numbers J KaKc to the upper and lower torsion–rotation levels involved in the observed transitions, relying heavily on comparison of quantum-beat patterns to determine transitions with a common upper state. These torsion–rotation transitions were then globally fit using a two-equivalent-top computer program, which was written in the principal axis system of the molecule and which uses a free-rotor basis set for each top, a symmetric-top basis set for the rotational functions, and a single-step diagonalization procedure. We can fit 411 lines involving 16 torsional sublevels from states with zero to three quanta of torsional excitation in the excited electronic state, using 24 parameters to obtain a standard deviation of 0.0045 cm −1, which is quite satisfactory, but inclusion in the fit of 440 transitions from all 17 rotationally assigned torsional levels increases the standard deviation by some 25%. The present fit gives a value of V 3 = 238 cm −1 for the threefold barrier height in the excited electronic state, in reasonable agreement with earlier studies.

Glucose Determination by Means of Steady-state and Time-course UV Fluorescence in Free or Immobilized Glucose Oxidase.

Changes in steady-state UV fluorescence emission from free or immobilizedglucose oxidase have been investigated as a function of glucose concentration.Immobilized GOD has been obtained by entrapment into a gelatine membrane. Changes insteady-state UV fluorescence have been quantitatively characterized by means ofoptokinetic parameters and their values have been compared with those previouslyobtained for FAD fluorescence in the visible range. The results confirmed that greatercalibration ranges are obtained from UV signals both for free and immobilized GOD inrespect to those obtained under visible fluorescence excitation. An alternative method tothe use UV fluorescence for glucose determination has been investigated by using timecourse measurements for monitoring the differential fluorescence of the redox forms of theFAD in GOD. Also in this case quantitative analysis have been carried out and acomparison with different experimental configurations has been performed. Time coarsemeasurements could be particularly useful for glucose monitoring in complex biologicalfluids in which the intrinsic UV fluorescence of GOD could be not specific by consideringthe presence of numerous proteins.

Photolysis of allene and propyne in the 7–30eV region probed by the visible fluorescence of their fragments

The photolysis of allene and propyne, two isomers of C(3)H(4), has been investigated in the excitation energy range of 7-30 eV using vacuum ultraviolet synchrotron radiation. The visible fluorescence excitation spectra of the excited neutral photofragments of both isomers were recorded within the same experimental conditions. Below the first ionization potential (IP), this fluorescence was too weak to be dispersed and possibly originated from C(2)H or CH(2) radicals. Above IP, three excited photofragments have been characterized by their dispersed emission spectra: the CH radical (A (2)Delta-X (2)Pi), the C(2) radical (d (3)Pi(g)-a (3)Pi(u), "Swan's bands"), and the H atom (4-2 and 3-2 Balmer lines). A detailed analysis of the integrated emission intensities allowed us to determine several apparition thresholds for these fragments, all of them being interpreted as rapid and barrierless dissociation processes on the excited potential energy surfaces. In the low energy range explored in this work, both isomers exhibit different intensity distributions in their fragment emission as a function of the photolysis energy, indicating that mutual allene<-->propyne isomerization is not fully completed before dissociation occurs. The effect of isomerization on the dissociation into excited fragments is present in the whole excitation energy range albeit less important in the 7-16 eV region; it gradually increases with increasing excitation energy. Above 19 eV, the fragment distribution is very similar for the two isomers.

Fluorescence studies of the excited-state proton transfer in substituted 3-hydroxychromones in supersonic jet

Abstract

Intramolecular excited-state proton transfer in jet-cooled 2-substituted 3-hydroxychromones and their water clusters

Intramolecular excited-state proton transfer (ESPT) of 3-hydroxychromone (3-HC), 3-hydroxyflavone (3-HF) and 2-(2-naphthyl)-3-hydroxychromone (2-NHC), and of their water clusters, was investigated in a supersonic expansion. The visible tautomer fluorescence excitation spectra of these compounds exhibit considerably well-resolved vibrational structures, while no significant uv fluorescence excitation spectrum due to the normal form was observed. The upper limit of the rate constant of the tautomer formation was estimated to be 1.77×1012 s−1 for 3-HC, 6.5×1011 s−1 for 3-HF, and 1.54×1011 s−1 for 2-NHC in supersonic expansion by simulation of the linewidth of the respective origin bands in the visible fluorescence excitation spectra. The order of rate constants of the tautomer formations in the supersonic free jet are consistent with that of the ESPT of these compounds in nonpolar solution. The fluorescence excitation and dispersed fluorescence spectra demonstrate 1:1 and 1:2 water complex formations of 3-HF and 2-NHC and no ESPT in these water clusters. However, no evidence of water complex formation was obtained in jet-cooled 3-HC.